Imaging of macular pigment distributions

ABSTRACT

Macular pigments are measured by spectrally selective lipofuscin detection. Light from a light source that emits light at a selected range of wavelengths that overlap the absorption band of macular carotenoids is directed onto macular tissue of an eye for which macular pigment levels are to be measured. Emitted light is then collected from the macular tissue. The collected light is filtered so that the collected light includes lipofuscin emission from the macular tissue at an excitation wavelength that lies outside the macular pigment absorption range and outside the excitation range of interfering fluorophores. The collected light is quantified at each of a plurality of locations in the macular tissue and the macular pigment levels in the macular tissue are determined from the differing lipofuscin emission intensities in the macula and peripheral retina.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/018,403, filed Dec. 21, 2004, entitled “Methods andApparatus for Detection of Carotenoids for Macular Tissue, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to techniques for optically measuringlevels of chemical compounds in biological tissues. More particularly,the invention relates to the noninvasive optical detection andmeasurement of levels of macular carotenoids and related chemicalsubstances using spectrally selective fluorescence spectroscopy oflipofuscin.

2. The Relevant Technology

Macular pigment (“MP”) is a collection of biological compoundsconcentrated in a small region in the center of the retina that provideshigh-acuity vision. Comprised of the carotenoid compounds lutein andzeaxanthin, MP is thought to play a protective role in the prevention ordelay of age-related macular degeneration (“AMD”), the leading cause ofirreversible blindness in the elderly in the Western world.Epidemiological studies analyzing carotenoid levels via dietary surveysand serum assays have shown that there is an inverse correlation betweenhigh dietary intakes and blood levels of lutein and zeaxanthin and riskof advanced AMD. Furthermore, several studies, including resonance Ramanclinical studies and a high performance liquid chromatography (“HPLC”)analysis of human cadaver eyes with and without a known history of AMD,have demonstrated a correlation between levels of lutein and zeaxanthinand AMD.

The standard methods that have been used for measuring carotenoids arethrough high-performance liquid chromatography (HPLC) techniques. Suchtechniques require that large amounts of tissue sample be removed fromthe patient for subsequent analysis and processing, which typicallytakes at least 24 hours to complete. In the course of these types ofanalyses, the tissue is damaged, if not completely destroyed. As aresult, there is a strong interest to develop non-invasive detectiontechniques for MP in the living human retina. Such techniques could beused, for example, in large-scale monitoring studies of dietary and/ornutritional interventions designed to raise MP levels, and potentiallyhelp protect a large fraction of the population from developing thisdebilitating disease.

Currently, the most commonly used noninvasive method for measuring humanMP levels is a subjective psychophysical heterochromatic flickerphotometry test involving color intensity matching of a light beam aimedat the fovea and another aimed at the perifoveal area. However, thismethod is rather time consuming and requires an alert, cooperativesubject with good visual acuity. This method can also exhibit a highintrasubject variability when macular pigment densities are low or ifsignificant macular pathology is present. Thus, the usefulness of thismethod for assessing macular pigment levels in the elderly populationmost at risk for AMD is severely limited. Nevertheless, researchers haveused flicker photometry to investigate important questions such asvariation of macular pigment density with age and diet.

A number of objective techniques for the measurement of MP in the humanretina have been explored recently as alternatives to the subjectivepsychophysical tests. The underlying optics principles of thesetechniques are either based on fundus reflection or fundus fluorescence(autofluorescence) spectroscopy.

One of the MP imaging approaches is based on fundus reflectancetechniques. There are two variants in which this method is implemented.In one variant, the reflectance of a broad band light source from thesclera is compared for a foveal and perifoveal spot and the spectralcontribution of the absorbance by MP is calculated. In a second variant,no reference at a peripheral site is needed. Only a foveal field isused, and MP levels are derived from a model fit that takes into accountthe absorption and scattering coefficients of all retinal layerstraversed in a double-path succession by the light.

Some reflectance based imaging variants are based on scanning laserophthalmoscopes (SLOs). Argon laser lines at 488 and 514 nm are used togenerate monochromatic digital reflection images from the retina at MP“on peak” and “off peak” spectral absorption positions, which are thendigitally subtracted to display the MP absorption distribution. Whilereflectance based MP imaging is evolving as a viable clinical techniquefor subjects, a drawback of the technology is seen in the need for eyesin mydriasis (dilation of the pupil).

In autofluorescence spectroscopy, lipofuscin in the retinal pigmentepithelium is excited with light within and outside the wavelength rangeof macular pigment absorption, but within the absorption range oflipofuscin. This can be realized, for example, with 488 nm and 532 nmlight sources, respectively. The blue (488 nm) wavelength is absorbedboth by macular pigment and lipofuscin; the green (532 nm) wavelength isabsorbed only by lipofuscin. By measuring the lipofuscin fluorescenceintensity levels for the foveal and peripheral retina regions, I_(min)and I_(max), respectively, for both excitation wavelengths, an estimateof the single-pass absorption of MP can be obtained. A disadvantage ofthe autofluoresecence technique is its low specificity. In principle,any absorber absorbing in the same wavelength range as the MP canartifactually attenuate the lipofuscin excitation, and thus lead to anerroneous mapping of the MP distribution and its concentration levels.This could be a serious drawback, particularly in the presence ofretinal pathology (e.g. drusen, bleeding vessels, etc). Similarly,fluorescence from other compounds than lipofuscin could confound theresults.

MP usually peaks in the center of the macula, the foveola, and drops offrapidly with increasing eccentricity. Absolute concentrations of MP arevery high compared to other tissue sites, corresponding typically to10-30 ng per macular punch biopsy (about 5 mm diameter). FIG. 1illustrates the absorption spectrum of an excised, flat-mounted, humanretina in the blue/green wavelength region, showing typical absorptioncharacteristics of carotenoid macular pigment (solid curve at left). Theretinal pigment epithelium of the retina was removed for thismeasurement, and the spectrum was measured through a 1 mm aperture. Inspite of the very thin retinal tissue layer, the optical density reachesan average value of about 0.3 above background, which explains theorigin of the strong yellow coloration of the macula. Comparing theoptical absorption of the macula with lutein and zeaxanthin solutions,one finds that the absorption behavior is remarkably similar, includingthe appearance of vibronic substructure, and that there is littleoverlap with potentially confounding other chromophores in the intactretina. The solid curve at right shows the fluorescence spectrum of asolution of lutein, obtained under excitation at 488 nm.

Optical excitation of MP leads to only very weak fluorescence since theexcited lutein and zeaxanthin molecules relax very rapidly (within200-250 fsec) to a lower lying excited state from which emission oflight is parity forbidden (see FIG. 1C). The unusual ordering of theenergy states is a unique feature of the polyene-like, π-conjugatedcarotenoids having a large number of conjugated C═C double bonds (10 and11 in lutein and zeaxanthin, respectively, see FIG. 1B). In fact, thequantum efficiency for a radiative transition from the 2 ¹Ag excitedstate to the 1 ¹Ag ground state is estimated to be as low as 10⁻⁵ to10⁻⁴. Therefore, relaxation of the excited molecule back to the groundstate occurs mostly via non-radiative transitions. The weak emission,observable only with very sensitive detection, has a small Stokes shift,and occurs in the green wavelength range centered at about 530 nm, asshown in FIG. 1 (solid curve at right).

Due to the weak fluorescence transitions, direct detection of MP usinglutein or zeaxanthin fluorescence has not been realized to date.However, the virtual absence of intrinsic MP fluorescence makes itpossible to detect instead the resonance Raman transitions of MP, evenin living human eyes, which would otherwise be masked beyond detectionby the fluorescence background. As a result, one method for themeasurement of carotenoids and related chemical substances in biologicaltissue is by resonance Raman spectroscopy, for example as disclosed inU.S. Pat. No. 6,205,354, the disclosure of which is incorporated byreference herein in its entirety. Generally, Raman spectroscopy is ahighly specific form of vibrational spectroscopy that identifies a Ramanshift, which corresponds to an energy which is the fingerprint of thevibrational or rotational energy state of certain molecules. Typically,a molecule exhibits several characteristic Raman active vibrational orrotational energy states, and the measurement of the molecule's Ramanspectrum thus provides a fingerprint of the molecule, i.e., it providesa molecule-specific series of spectrally sharp vibration or rotationpeaks. The intensity of the Raman scattered light corresponds directlyto the concentration of the molecule(s) of interest. In the case ofRaman spectroscopy as applied to MP, this method detects the light thatis Raman scattered from the MP carotenoid molecules at their 1525 cm⁻¹carbon-carbon double bond stretch frequency under resonant excitation inthe MP absorption band. The Raman method measures the response of MPdirectly and has a very high molecule specificity.

Raman detection methods have the benefit of extremely high specificityfor lutein and zeaxanthin, and therefore for MP. However, detecting onlythe absolute amount of MP, the Raman response is attenuated to somedegree by the combined absorption and scattering of anterior ocularmedia, predominantly the lens.

Accordingly, improved methods and apparatus that quickly, safely, andaccurately measure a human's macular carotenoid levels are needed.

BRIEF SUMMARY OF THE INVENTION

According to the invention, macular pigment (“MP”) imaging based onlipofuscin fluorescence detection is useful as a relatively simple,objective and quantitative noninvasive optical technique suitable torapidly screen MP levels and distributions in healthy humans withundilated pupils. The invention can be used in a direct and quantitativeoptical diagnostic technique, which uses low intensity illumination ofintact tissue and provides high spatial resolution, allowing for precisequantification of the carotenoid levels in the tissue.

The detection of lipofuscin fluorescence, in combination with suitablesignal processing, can be used to indirectly derive the concentrationsand spatial distributions of MP in living human subjects. Particularly,the invention enables the elimination of the confounding effects offluorophores, found mainly in the lens, in detected intensity maps. Theconfounding effect on the optically detected MP levels can be avoided byusing a transmission filter in the fluorescence detection channel thatlimits the detection to wavelengths on the long-wavelength shoulder ofthe lipofuscin fluorescence spectrum. Using a direct, CCD basedfluorescence imaging setup with suitable light excitation sources, likelasers, light emitting diodes (LEDs), or conventional light sources withsuitable filters, and filtered signal detection, MP distributions can bemeasured within a fraction of a second through undilated eyes with highreproducibility.

The spatial and quantitative information of retinal MP levelssimultaneously measurable with the lipofuscin-based technique withoutpupil dilation can be expected to be of tremendous value in screening oflarge populations. It can be a tool in studies investigating theinfluence of dietary supplements on MP levels and their distributions,and in research investigating the link between MP levels and retinalpathologies. Since the fluorescence based MP detection method is notinfluenced, in first order, by anterior media opacities, it could have aparticular advantage over the Raman method in elderly subjects, which ingeneral have higher lens opacities, and often reduced MP levels.

Accordingly, a first example embodiment of the invention is a method formeasuring macular pigments. The method generally includes firstproviding a light source that emits light at a selected range ofwavelengths that simultaneously overlaps the absorption band of macularcarotenoids and the absorption band of lipofuscin. The light is directedfrom the light source onto macular tissue of an eye for which macularpigment levels are to be measured, light emitted from the macular tissueis collected, the collected light comprising lipofuscin emission fromthe macular tissue at an excitation wavelength that lies outside themacular pigment absorption range and outside the excitation range ofinterfering fluorophores. The collected light is quantified for each ofa plurality of locations in the macular tissue and the lipofuscinemission intensities from each of a plurality of locations is therebydetermined as well. Finally, the macular pigment levels in the maculartissue are determined from the differing lipofuscin emission intensitiesin the macula and peripheral retina. Further details and variants onthis method are described elsewhere herein.

Another example method of the invention is also a method for measuringmacular pigments. This method generally includes: providing a lightsource that emits light at a selected range of wavelengths that overlapthe absorption band of macular carotenoids; directing light from thelight source at an intensity of less than about 10 mJ/cm² for less than500 msec onto macular tissue of an undilated eye for which macularpigment levels are to be measured; collecting light emitted from themacular tissue, the collected light comprising lipofuscin emission fromthe macular tissue at an excitation wavelength that lies outside themacular pigment absorption range and outside the excitation range ofinterfering fluorophores in the lens; quantifying the collected lightfrom each of a plurality of locations in the macular tissue and therebyquantifying the lipofuscin emission intensities from each of a pluralityof locations; determining the macular pigment levels in the maculartissue from the differing lipofuscin emission intensities in the maculaand peripheral retina; and determining spatial extent and topographicconcentration distribution of the macular pigments.

Yet another non limiting example embodiment of the invention is anapparatus for measuring macular pigments. The apparatus includes a lightsource that generates light at a wavelength that produces anautofluorescence lipofuscin emission. One or more light delivery opticalcomponents such as fibers, a shutter, lens filters, etc. are used fordirecting light from the light source to a subject's eye. Lightcollection optical components are used for receiving an autofluorescencelipofuscin emission from the subject's eye and routing theautofluorescence lipofuscin emission from the eye. An optical filter isconfigured for receiving the autofluorescence lipofuscin emission and isselective for passing light at a selected wavelength range such thatfluorescence contributions from ocular media besides the macula aresubstantially blocked. An optical detector is configured for receivingthe passed light from the optical filter and generating a signalindicative of the fluorescence intensities of the lipofuscin emission ata plurality of points on the subject's eye. A computing device can beused to determine intensities of the lipofuscin emission from the passedlight at a plurality of points.

These and other features of the present invention will become more fullyapparent from the following description and appended claims, or may belearned by the practice of the invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 a illustrates the absorption spectrum of an excised,flat-mounted, human retina in the blue/green wavelength region, showingtypical absorption characteristics of carotenoid macular pigment (solidcurve at left) and the very weak fluorescence of macular pigment (solidcurve at right) obtained when exciting them in their absorption band;

FIG. 1 b illustrates the molecular structure of lutein and zeaxanthin;

FIG. 1 c is an energy level diagram of long-chain conjugated carotenoidslike lutein or zeaxanthin, with optical and non-radiative transitionslike excitation, Raman, and fluorescence, indicated as arrows;

FIG. 2 a illustrates the absorption and emission spectra of a methanolicsolution of A2E, the main fluorophore of lipofuscin, shown as solidcurves at left and right side of panel, respectively. The absorptionspectrum of macular pigment is indicated as a dashed curve;

FIG. 2 b illustrates molecular structures of A2E (left) and iso-A2E;

FIG. 2 c is an energy level diagram for lipofuscin with opticaltransitions shown as arrows;

FIG. 3 a is a schematic representation of retinal layers participatingin light absorption, transmission, and scattering of excitation andemission light in the macular region;

FIG. 3 b is the schematics of anterior optical media and retinal layerstraversed by excitation laser light, fluorescence from anterior opticalmedia and fluorescence from lipofuscin;

FIG. 4 illustrates schematics of an apparatus used for imaging MPdistributions in living human subjects according to the invention;

FIG. 5 illustrates the removal of laser speckle effects in fluorescenceimaging;

FIG. 6 illustrates the determination of a spatial correction factor forimage processing due to spatially varying intensity profiles in laserexcitation and lipofuscin fluorescence disks;

FIG. 7 is a white-light fundus camera image of a healthy subject (imagea), and lipofuscin fluorescence images of the fundus obtained at nearinfrared wavelengths with 488 nm (image b) and 532 mn (image c) laserexcitation, respectively;

FIG. 8 illustrates schematics showing processing of CCD pixel intensityregions to derive optical density values of MP absorption at any desiredlocation in the retina;

FIG. 9 a is a top view schematic illustration of a setup used in themeasurement of lutein concentrations in a tissue phantom consisting of adried lutein spot located on the side wall of a thin cuvette filled withan optically thin A2E solution (O.D. about 0.35);

FIG. 9 b is a side view schematic illustration of a setup used in themeasurement of lutein concentrations in a tissue phantom consisting of adried lutein spot located on the side wall of a thin cuvette filled withan optically thin A2E solution (O.D. about 0.35);

FIG. 9 c is a plot illustrating optical densities of luteinconcentrations measured for several dozen positions within the driedlutein spot, using simultaneous lipofuscin fluorescence attenuation andtransmission measurements of the individual spots, showing excellentcorrelation (correlation coefficient R=0.96);

FIG. 10 illustrates the wavelength dependence of MP optical densityobtained when successively limiting the detected fluorescence range tolonger wavelength, using long-wavelength pass filters with suitablecut-on wavelengths, λc;

FIG. 11 displays lipofuscin fluorescence images of four volunteersubjects (a-d), obtained under 488 nm excitation, with intensity levelscoded in gray scale;

FIG. 12 shows lipofuscin fluorescence intensity profiles, derived fromFIG. 11, along nasal-temporal meridans (solid curves) andinferior-superior meridians (dashed curves), all running through thecenter of the macula (see insert);

FIG. 13 shows MP optical density plot profiles along the nasal-temporalmeridian, derived from FIG. 11 for each subject;

FIG. 14 shows pseudo-color scaled, 3-d, MP distributions derived fromthe 2-d lipofuscin fluorescence pixel intensity maps of FIG. 11;

FIG. 15 illustrates the effect of blood vessels on transmission lineplots derived from gray-scale lipofuscin fluorescence intensity maps;

FIG. 16 a illustrates categories of MP distributions observed inclinical measurements of 70 subjects (total of 122 eyes);

FIG. 16 b illustrates the distribution of 122 measured eyes amongcategories A to E. A large fraction of subjects, 28%, has a sharp,central MP distribution;

FIG. 17 illustrates the macular pigment levels measured in 90 eyes of aclinical population of 70 volunteer subjects, displayed as a function ofage. Each data point corresponds to the maximum MP level determined fromlipofuscin fluorescence images; and

FIG. 18 illustrates the correlation between MP levels determined bylipofuscin fluorescence based imaging and resonance Raman spectroscopy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the invention, lipofuscin fluorescence spectroscopy(“autofluorescence or AF spectroscopy”) can be used to noninvasively andindirectly derive the concentrations and spatial distributions of MP inliving mammals, e.g. human subjects. Conventional approaches suffer fromthe effects of fluorophores in the living human lens that havepreviously confounded lipofuscin fluorescence techniques. Thefluorophores in the living human lens are simultaneously excited withthe lipofuscin molecules to generate a strong fluorescence in thegreen/yellow wavelength range. According to one aspect of the invention,the confounding effect on the optically detected MP levels can beavoided by using a transmission filter in the fluorescence detectionchannel that limits the detection to wavelengths on the long-wavelengthshoulder of the lipofuscin fluorescence spectrum. For example, using adirect, CCD based fluorescence imaging setup with laser excitation andfiltered signal detection, the invention makes it possible to measure MPdistributions within a fraction of a second with high reproducibility,while avoiding confounding effects of lens fluorescence.

Embodiments of the invention can be performed or used quickly innon-mydriatic conditions. A mydriatic is an agent which induces dilationof the pupil. Normally, mydriatic drugs such as tropicamide are used inophthalmology to permit examination of the retina and other deepstructures of the eye. Applying a mydriatic to examine the retina isinherently risky because it removes a protective defense of the body.The non-mydriatic uses of the invention can therefore provide asignificant advantage by increasing the safety, speed, and frequencywith which the tests can be performed.

Reference will now be made to the drawings to describe various aspectsof exemplary embodiments of the invention. It is to be understood thatthe drawings are diagrammatic and schematic representations of suchexemplary embodiments, and are not limiting of the present invention,nor are they necessarily drawn to scale.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present invention. Itwill be obvious, however, to one skilled in the art that the presentinvention may be practiced without these specific details. In otherinstances, well-known aspects of the molecules and compositionsdiscussed herein, the physics of light, and optical systems in generalhave not been described in particular detail in order to avoidunnecessarily obscuring the present invention.

Human retinal pigment epithelium contains the age pigment lipofuscinwhich accumulates in the lysosomal body of the RPE cells. Two componentsof lipofuscin exist that absorb strongly in the blue wavelength regionand emit strongly in the orange-red region. They are two isomers of abis-substituted pyridinium ring, and are termed A2E and iso-A2E,respectively. The structure of the molecules, their optical transitions,and their associated energy level scheme are shown in FIGS. 2 a, 2 b,and 2 c.

The absorption spectrum of A2E partially overlaps with the absorptionspectrum of the macular carotenoids, and its emission spectrum is broad,extending to wavelengths well beyond the absorption of carotenoids, asshown in FIG. 2 a. Particularly, FIG. 2 a is a graph of the absorption(solid curve at left) and emission spectra (solid curve at right) ofA2E, the main fluorophore of lipofuscin, dissolved in methanol. Theabsorption peaks in the blue spectral range at about 430 nm, and theemission in the red spectral range at about 640 nm. The absorption ofthe macular pigments lutein and zeaxanthin is also indicated, as adotted line, and shows that it essentially occurs in the same spectralrange as that of lipofuscin, with the important exception thatlipofuscin absorbs also to longer wavelengths compared to the macularpigments, so that a wavelength range exists on the long-wavelengthshoulder of the lipofuscin absorption band where lipofuscin can excitedexclusively without being attenuated by the macular pigment absorption.Two spectral positions of laser excitation lines, 488 nm and 532 nm,respectively, are shown as arrows. The 488 nm line is seen to overlapboth the lipofuscin and the MP absorption on the long wavelengthshoulder. The 532 nm line is outside the spectral absorption range of MPbut overlaps that of lipofuscin.

It is possible, therefore, to excite the A2E emission within and outsidethe absorption range of MP. Similar to lutein and zeaxanthin, A2E has aconjugated carbon backbone. However, the conjugation length is shorter,interrupted by the central pyridinium ring, which shortens theconjugated carbon bond chain to two smaller chains with five doublebonds each. This leads to an emission with a quantum efficiency of 10⁻²,which is about three orders of magnitude stronger than that reported forlutein and zeaxanthin. The in-vivo excitation spectrum of fundusautofluorescence is much broader than that of A2E, suggesting that otherlipofuscin fluorophores contribute to fundus autofluorescence. Regardingits spatial distribution, it is known that the lipofuscin fluorescenceat 15° from the fovea is 1.4-1.7 times higher than at the fovea and thatthere is a gradual increase in lipofuscin fluorescence with increasingeccentricity.

As will be described in greater detail elsewhere herein, the shading tothe right of about 700 nm indicates the wavelength range where along-wavelength pass filter used for the measurement of lipofuscinemission has reached transparency, limiting the detection of thelipofuscin emission intentionally only to wavelengths beyond about 700nm.

With reference to FIG. 3 a, excitation light, indicated by shadedarrows, is shown reaching a retina, where the MP levels are generallydepicted as shaded areas. In MP Raman detection, which usesbackscattering of excitation light from the MP-containing Henle fiberlayer, detection is not influenced by deeper retinal layers as indicatedby the limited depth of the filled arrows. In contrast, emission oflipofuscin used in autofluorescence-based measurements of MP has totraverse the photoreceptor (PhR) layer, and also deeper layers of theretina. Therefore, it has to take into account additional absorption andemission effects of these layers and their fluorophores.

As seen in resonance Raman detection experiments of MP, that detect thespectrally sharp C═C double bond 1525 cm⁻¹ stretching vibration Ramanresponse of lutein and zeaxanthin (M), which occur at 527 nm under 488nm excitation, a strong fluorescence background is superimposed on theRaman response in the 530 nm region. This fluorescence background variesstrongly from subject to subject and exceeds in strength the backgroundlevel expected from intrinsic lutein or zeaxanthin (MP) fluorescence.This background is a combination of lipofuscin fluorescence andfluorescence from anterior ocular media. In Raman detection, the broadfluorescence background can be easily fitted with a higher orderpolynomial and subtracted from the measured spectra.

In lipofuscin-based MP measurements, however, the extra fluorescenceresponses can be expected to play an interfering role. Methods ofhandling these extra fluorescence responses are therefore describedbelow.

The optical layers of interest traversed by the excitation light and thelipofuscin fluorescence are sketched in FIG. 3 b. The fluorescenceintensity in 180 degree detection geometry, I_(Det), measured at aselected wavelength, λ, in the visible/near IR wavelength range, is ingeneral given byI _(Det)(λ)=I _(L)(λ)·T_(PR)(λ)·T_(MP)(λ)·T_(OM)(λ)+I _(OM)(λ),   (Eq.1)where T(λ) is the transmission of the respective layer corresponding tophotoreceptors, PR, macular pigment, MP, and anterior ocular media, OM,at the fluorescence wavelength, λ. I_(L) is the lipofuscin fluorescenceoriginating in the fovea, and I_(OM)(λ) is the potentially overlappingfluorescence intensity of the OM layer at λ.

It is safe to assume that the photoreceptors do not generate anyfluorescence, but their absorption can not be neglected, in general.Under excitation with I_(exc) at wavelength λ_(exc), the lipofuscinfluorescence intensity I_(L)(λ) is correlated with I_(exc) according toI _(L)(λ_(exc),λ)=η_(L)(λ_(exc),λ)·I _(exc)(λ_(exc))·T _(OM)(λ_(exc))·T_(MP)(λ_(exc))·T _(PR)(λ),   (Eq. 2)and I_(OM) is correlated with I_(exc) according toI _(OM)(λ_(exc),λ)=η_(OM)(λ_(exc),λ)·I _(exc)(λ_(exc))·(1−T _(OM)),  (Eq. 3)where η_(L)(λ,λ_(exc)) and η_(OM)(λ,λ_(exc)) are the fluorescencequantum efficiencies, respectively, of lipofuscin and the anteriorocular media.

Inserting equations (2) and (3) into (1), one obtains $\begin{matrix}{{I_{Det}\left( {\lambda_{exc},\lambda} \right)} = {{{\eta_{MP}\left( {\lambda_{exc},\lambda} \right)} \cdot {I_{exc}\left( \lambda_{exc} \right)} \cdot {T_{OM}\left( \lambda_{exc} \right)} \cdot {T_{MP}\left( \lambda_{exc} \right)} \cdot {T_{PR}\left( \lambda_{exc} \right)} \cdot {T_{OM}(\lambda)} \cdot {T_{MP}(\lambda)} \cdot {T_{PR}(\lambda)}} + {{\eta_{OM}\left( {\lambda_{exc},\lambda} \right)} \cdot {I_{exc}\left( \lambda_{exc} \right)} \cdot {\left\lbrack {1 - {T_{OM}(\lambda)}} \right\rbrack.}}}} & \left( {{Eq}.\quad 4} \right)\end{matrix}$

In general, it is impossible to extract the isolated MP transmission(absorption) from this complex relation between detector intensity andtransmission/fluorescence terms of all layers. However, adopting astring of assumptions and approximations, it becomes possible toeliminate, in first order, several unwanted terms in (4), and to derivean approximation for the MP transmission (absorption) difference betweenfovea and perifovea.

First, as substantiated below (section 3), it is possible to largelyblock the detection of OM fluorescence by limiting the detection oflipofuscin fluorescence to near IR wavelengths. Only under thiscondition can the second term in (Eq. 4) can be ignored. Second, it isuseful to reference the detected luminescence in the fovea to ameasurement in the perifovea, and to determine this ratio for twoexcitation wavelengths λ₁ and λ₂ near the peak and just outside the MPabsorption band, respectively. For the ratios of the MP transmissions inperifovea and fovea at the two wavelengths one then obtains$\begin{matrix}\begin{matrix}{\frac{I_{Det}^{P}\left( {\lambda_{exc},\lambda} \right)}{I_{Det}^{F}\left( {\lambda_{exc},\lambda} \right)} = {\frac{\eta_{MP}^{P}\left( {\lambda_{exc},\lambda} \right)}{\eta_{MP}^{F}\left( {\lambda_{exc},\lambda} \right)} \cdot \frac{{T_{OM}^{P}\left( \lambda_{exc} \right)}{T_{OM}^{P}(\lambda)}}{{T_{OM}^{F}\left( \lambda_{exc} \right)}{T_{OM}^{F}(\lambda)}} \cdot}} \\{\frac{{T_{MP}^{P}\left( \lambda_{exc} \right)}{T_{MP}^{P}(\lambda)}}{{T_{MP}^{F}\left( \lambda_{exc} \right)}{T_{MP}^{F}(\lambda)}} \cdot \frac{{T_{PR}^{P}\left( \lambda_{exc} \right)}{T_{PR}^{P}(\lambda)}}{{T_{PR}^{F}\left( \lambda_{exc} \right)}{T_{PR}^{F}(\lambda)}}}\end{matrix} & \left( {{Eq}.\quad 5} \right)\end{matrix}$If one assumes that the lipofuscin composition is constant across theposterior pole, the quantum efficiency ratio term$\left( {\frac{\eta_{MP}^{P}\left( {\lambda_{1},\lambda} \right)}{\eta_{MP}^{F}\left( {\lambda_{1},\lambda} \right)}/\frac{\eta_{MP}^{P}\left( {\lambda_{2},\lambda} \right)}{\eta_{MP}^{F}\left( {\lambda_{2},\lambda} \right)}} \right)$is eliminated. If one further assumes that there is no differencebetween foveal and perifoveal lens transmission terms cancel out.Furthermore, if one insures that the excitation light beam profiles areidentical for the two wavelengths, they cancel out, too. One thenobtains the much simplified expression $\begin{matrix}\begin{matrix}{{\left( \frac{I_{Det}^{P}\left( {\lambda_{1},\lambda} \right)}{I_{Det}^{F}\left( {\lambda_{1},\lambda} \right)} \right)/\left( \frac{I_{Det}^{P}\left( {\lambda_{2},\lambda} \right)}{I_{Det}^{F}\left( {\lambda_{2},\lambda} \right)} \right)} = {\left( {\frac{T_{MP}^{P}\left( \lambda_{1} \right)}{T_{MP}^{F}\left( \lambda_{1} \right)}/\frac{T_{MP}^{P}\left( \lambda_{2} \right)}{T_{MP}^{F}\left( \lambda_{2} \right)}} \right) \cdot}} \\{\left( {\frac{T_{PR}^{P}\left( \lambda_{1} \right)}{T_{PR}^{F}\left( \lambda_{1} \right)}/\frac{T_{PR}^{P}\left( \lambda_{2} \right)}{T_{PR}^{F}\left( \lambda_{2} \right)}} \right).}\end{matrix} & \left( {{Eq}.\quad 6} \right)\end{matrix}$

Referencing the transmissions of the excitation light at the λ₁ and λ₂positions to the transmission values in the peak of the MP absorptionband, 460 nm, with respective extinction coefficients K_(MP)(λ₁) andK_(MP)(λ₂), whereT _(MP) ^(P,F)(λ₁)=T _(MP) ^(P,F)(460)^(K(λ) ¹ ⁾ , T _(MP) ^(P,F)(λ₂)=T_(MP) ^(P,F)(460)^(K(λ) ² ⁾,the right side of (Eq. 6) reduces to [T_(MP) ^(P)(460)/T_(MP)^(F)(460)]^([K) ^(MP) ^((λ) ¹ ^()−K) ^(MP) ^((λ) ² ^()]). With O.D.=log(1/T), and solving for the MP optical density difference, this can bewritten as $\begin{matrix}\begin{matrix}{\begin{matrix}{{O.D._{MP}^{\quad F}\left( \lambda_{\quad 460} \right)} -} \\{O.D._{MP}^{\quad P}\left( \lambda_{\quad 460} \right)}\end{matrix} = {\frac{1}{\Delta\quad K}\left\{ {{\log\left( \frac{I_{Det}^{P}\left( {\lambda_{1},\lambda} \right)}{I_{Det}^{F}\left( {\lambda_{1},\lambda} \right)} \right)} -} \right.}} \\{\left. {\log\left( \frac{\quad{I_{\quad{Det}}^{\quad P}\quad\left( \quad{\lambda_{\quad 2},\quad\lambda} \right)}}{\quad{I_{\quad{Det}}^{\quad F}\quad\left( \quad{\lambda_{\quad 2},\quad\lambda} \right)}} \right)} \right\} -} \\{\frac{1}{\quad{\Delta\quad K}}{{\log\left( {\frac{T_{PR}^{P}\left( \lambda_{1} \right)}{T_{PR}^{F}\left( \lambda_{1} \right)}/\frac{T_{PR}^{P}\left( \lambda_{2} \right)}{T_{PR}^{F}\left( \lambda_{2} \right)}} \right)}.}}\end{matrix} & \left( {{Eq}.\quad 7} \right)\end{matrix}$

If one assumes in addition that the photoreceptors can be completelybleached in the measurements (T_(PR)=100%), the second term can beneglected. Then the MP optical density difference between fovea andperifovea is given by $\begin{matrix}\begin{matrix}{\begin{matrix}{{O.D._{MP}^{\quad F}\left( \lambda_{\quad 460} \right)} -} \\{O.D._{MP}^{\quad P}\left( \lambda_{\quad 460} \right)}\end{matrix} = {\frac{1}{\Delta\quad K}\left\{ {{\log\left( \frac{I_{Det}^{P}\left( {\lambda_{1},\lambda} \right)}{I_{Det}^{F}\left( {\lambda_{1},\lambda} \right)} \right)} -} \right.}} \\{\left. {\log\left( \frac{\quad{I_{\quad{Det}}^{\quad P}\quad\left( \quad{\lambda_{\quad 2},\quad\lambda} \right)}}{\quad{I_{\quad{Det}}^{\quad F}\quad\left( \quad{\lambda_{\quad 2},\quad\lambda} \right)}} \right)} \right\},}\end{matrix} & \left( {{Eq}.\quad 8} \right)\end{matrix}$i.e. it can be approximated by the log differences of foveal andperifoveal fluorescence intensities.

It should be mentioned that besides lipofuscin detection at nearinfrared wavelength other approaches are possible to minimize theeffects of media fluorescence. These include confocal optics, separationof excitation and detection beams through the lens, and correction forlens fluorescence.

It is instructive to compare the derived expressions with the expressionfor the MP signal intensity obtained with the Raman method. Since theRaman response originates directly in the MP layer, deeper layers do notplay a role in the excitation and emission paths. Only the anterioroptical media are traversed, resulting in $\begin{matrix}{I_{S} = {{{T_{OM}\left( \lambda_{exc} \right)} \cdot {T_{OM}\left( \lambda_{Raman} \right)} \cdot {N\left( E_{I} \right)} \cdot \sigma_{R} \cdot {I_{exc}\left( \lambda_{exc} \right)}} + {{\eta_{OM}\left( {\lambda_{exc},\lambda_{Raman}} \right)} \cdot {I_{exc}\left( \lambda_{exc} \right)} \cdot {\left\lbrack {1 - {T_{OM}\left( \lambda_{Raman} \right)}} \right\rbrack.}}}} & \left( {{Eq}.\quad 9} \right)\end{matrix}$

Here, I_(S) is the Raman scattered light intensity, N(E_(I)) is theconcentration of MP molecules, σ_(R) is the Raman scattering crosssection, and I_(exc) is the laser excitation intensity. No assumptionsregarding photoreceptor bleaching have to be made, and since thefluorescence of the lens is easily distinguishable from the spectrallysharp Raman response of MP, it can be easily subtracted. On the otherhand, since very little MP exists outside the macula area, foveal Ramanmeasurements can not be referenced to perifoveal levels, and lensabsorptions as well as all anterior media scattering will reduce theobserved Raman intensities.

An example of an inventive apparatus 100 where it can be employed formeasuring macular pigments using autofluorescence spectroscopy, isschematically depicted in FIG. 4. This system is configured forobtaining MP measurements by direct, large diameter, laser or non-laserlight excitation of a foveal and peripheral macular area, spectrallyisolating the captured fluorescence, and using an imaging CCD camera forfluorescence detection. As described below, the system is designed tomaximize the throughput for fluorescence originating from the retina.

The depicted apparatus 100 includes a light source(s) that emits lightat a wavelength or wavelength range that overlaps the absorption band ofboth macular carotenoids and the absorption band of lipofuscin, forexample a first coherent, or non-coherent conventional light source 102and an optional second coherent light source 104, for example 488 nm and532 nm fiber-coupled solid state lasers. Alternatively, light sources102 and 104 may comprise other devices, such as light emitting diodes(LEDs) for generating nearly monochromatic light, or suitably filtered,i.e. spectrally narrowed, conventional light sources. It is because ofthe other inventive aspects of the system and methods described hereinthat relatively low power and low cost LEDs can be used.

The light sources 102 and 104 are in optical communication with one ormore light delivery optical components, which direct laser light to themacular tissue to be measured. The laser beams are combined in anoptical beam combining cube 106, sent into an optical fiber 122,expanded at the output end of fiber 122 with an achromatic lens 124, andfiltered by a narrow-band interference filter 126. The light is thenprojected onto the retina using focusing lens 128, a dichroicholographic beam splitter 30 and the combination of the human cornea andeye lens 133 (eye lens is not shown). The resulting excitation disk onthe retina is preferably about 3.5 mm in diameter. The excitation diskhas an intermediate focus at the position of an aperture 132 that ispositioned in front of the eye (about 1 cm distance), and thateffectively blocks spurious reflections originating from beam splitter130.

To ensure steady fixation of the eye during measurements, a red aiminglaser 136 is used as the fixation target. It is routed into the setupwith an uncoated quartz beam splitter 138. The fixation light isdirected either to the fovea, for measurements centered on the fovea ofthe macular region, or to the peripheral macula (about 7 degreeseccentricity). The fixation laser beam diameter on the retina is about200 microns. The optical shutter 120 is designed such that it transmitsa small portion of the excitation light even when it is closed. Thisallows the subject to view both the red fixation target and thesuperimposed laser excitation disk for optimum head alignment, which isfurther facilitated by an adjustable chin rest.

At prolonged viewing, the leaked excitation light (6.9 log trolands)effectively bleaches more than 90% of the photoreceptor pigments of themacula. This establishes uniform background absorption throughout theexposed retina.

The lipofuscin fluorescence is captured by one or more light collectionoptical components, which direct laser light to the macular tissue to bemeasured. As depicted in FIG. 4, light is thus routed back through beamsplitter 130, which is transparent for wavelengths above about 580 nm,through holographic notch filter 140, which blocks excitation lightwavelengths, and through a long-pass filter, 142, which is transmissiveabove 700 nm. Limitation of the fluorescence to wavelengths above 700 nminsures rejection of light from unwanted fluorophores like the lens, andprovides optimum contrast between peripheral and macular fluorescenceintensities, as shown below.

The light beam delivery system is in optical communication with a anoptical detector such as a light detection system 146, that is used tomeasure the intensity of the scattered light over the target area ofinterest. The light detection system 146 may include, but is not limitedto, devices such as a CCD (charge coupled device) camera or detectorarray, an intensified CCD detector array, a photomultiplier apparatus,photodiodes, or the like. For example, the light can be collected by a50 mm focal length achromat 144 and imaged onto the pixel array of a CCDcamera (e.g. a 512×512 pixel array of a CCD camera Model ST-9 XE,available from the Santa Barbara Instrument Group, Inc.). For the ModelST-9 XE camera, individual pixel dimensions are 20 micron height by 20micron width; quantum efficiency in the red/near IR drops slowly from60% at 650 nm to 40% at 800.

The detected light is converted by light detection system 146 into anelectronic signal, typically processed by software associated with thedetection system, and sent to a computing device such as amicroprocessor or personal computer 142 or the like. The computer 148can control the shutter 120 as well. For data analysis, the pixelintensity maps can be converted to proportional files, and furtherprocessed using suitable image processing software (available, e.g. fromThe MathWorks, Inc.).

The signal is then analyzed and visually displayed on the monitor ofcomputer 148. It should be understood that the light detection system146 may also convert the light signal into other digital or numericalformats, if desired. The resultant signal intensities may be calibratedby comparison with chemically measured carotenoid levels from otherexperiments. The computer 148 preferably has data acquisition softwareinstalled that is capable of spectral manipulations.

During operation of apparatus 100, laser excitation light from eitherlight source 102 or 104 is routed via optical beam combining cube 106,mechanical shutter 120, optical fiber 122, and dichroic beam splitter130 to the retina of the eye to be measured. The lenses 124 and 128image the output face of the optical fiber delivering the laserexcitation light onto the retina of the eye to be measured. The notchfilter 126 transmits only the laser excitation light. The lipofuscinemission from the retina of the measured eye is transmitted by dichroicbeam splitters 130 and 138 and detected by light detection system, 146such as a CCD camera, after traversing filter 140, 142 and lens 144. Ared aiming light 136, serving as a fixation target during themeasurement, is projected onto the retina of the eye via dichroic beamsplitter 138. The pass filter 142 transmits only the long-wavelengthemission of lipofuscin (e.g., at wavelengths larger than about 700). Thelight detection system 146 then converts the signal into a form suitablefor visual display such as on a computer monitor or the like. Forexample, spatial digital MP images of a subject are recorded bydetecting the lipofuscin fluorescence of the retinal pigment epitheliumin its long-wavelength emission range upon sequential excitation with488 nm and 532 nm light, and the spatial extent of MP and itstopographic concentration distribution is obtained by digital imageprocessing according to equation (8), which under these wavelengthconditions reduces to equation (10), shown below.

Preferably, apparatus 100 is operated on a living mammal, preferable aperson, with undilated eyes in a darkened room. Background noise levelscan be in the range of 100 counts per pixel; signal counts typicallyabout 30,000 counts per pixel. The CCD camera temperature is preferablykept uniform, for example at a temperature at or below 5° C. duringmeasurements.

Laser speckle in the images obtained when using coherent laser light asexcitation sources can be effectively removed by mechanically shakingthe light delivery fiber during measurements, which generates aspatially homogeneous laser excitation spot via fiber mode mixing. Theresulting speckle removal effect and its impact on the obtainable imagequality is illustrated in FIG. 5 for images of millimeter graph paper,(a) to (b), and the macular area of a human retina (c) to (d),respectively, demonstrating that fine details such as small retinalblood vessels in the peripheral macula can be resolved after speckleremoval. Since a 1 mm distance is imaged onto a pixel array distance of120 pixels, the invention provides about a 10 micron spatial resolutionunder the used imaging conditions after speckle removal. Speckle removalprocedures will not be necessary if incoherent, non-laser lightexcitation sources are used.

For correct imaging of large tissue regions, and for the purpose ofcomparing MP measurements for single-wavelength (488 nm) anddual-wavelength (488 and 532 nm) measurements, intensity correctionshave to be made that account for intensity variations of the typicallyGaussian shaped laser beam profile and the resulting fluorescenceresponse across the excitation spot. To illustrate this point, thelipofuscin distribution in the peripheral macula and in the foveal areawas imaged using an approximately 3.5 mm diameter light excitation disk.As seen from FIG. 6, the measured lipofuscin intensity distribution,which is shown as dashed curve, follows again a Gaussian beam profile,dropping for both wavelengths (488 and 532 nm) by about 20% from thecentral intensity level to reduced level at the periphery of thefluorescence image. Thus, fluorescence intensity levels at the edge ofthe image disk have to be multiplied by a corresponding correctionfactor (about 25%) before comparing them with central image values.

To eliminate the potential for any deviations from these profiles inliving human eyes due to scattering and absorption effects of theanterior ocular media, the lipofuscin distribution of the retina wasimaged in two specific locations. As sketched in FIG. 6, one of the twoimages is centered on the fovea. It is recorded for this centrallyfixated subject such that a 7-degree eccentric location, termed S, isrecorded along with the fovea, F. Subsequently, a second image isrecorded which is now centered on the same peripheral location S,achieved by having the subject fixate on the aiming laser that has beenchanged in direction by 7 degrees. Since the spot S has an unchanginglipofuscin concentration, any detected difference in fluorescenceintensities originating from the spot S in the two images has to be dueto the laser excitation intensity beam profile plus the combination ofscattering and absorption effects of anterior ocular media. Comparingthe corresponding intensities of the two images, in view of thedisclosure herein one can derive a spatial correction factor map foreach subject.

In a preferred aspect of the invention, laser power levels at the corneacan be 2 mW during measurements with exposure times of 200 msec. At aretinal excitation disk size of 3.5 mm diameter, the light exposure wasthus about 3 mJ/cm², which is approximately a factor of 3 below thephotothermal safety limit of 10 mJ/cm² set forth by the ANSI standard.The photochemical limit for retinal injury is listed in the samestandard as 15.5 J/cm² for the used wavelengths. At the used energydensity of 3 mJ/cm², the exposure is therefore a factor of about 5,000below the photochemical limit. Accordingly, exposures suitable for usein the invention are preferably less than about 10 mJ/cm², morepreferably less than about 5 mJ/cm², still more preferably less thanabout 3 mJ/cm². Exposure times are preferably less than about 1 second,more preferably less than about 500 msec, still more preferably lessthan about 200 msec. In fact, and as mentioned elsewhere herein, suchlow levels can be used with the methods and apparatus of the inventionthat light emitting diodes can substitute for lasers in someembodiments.

FIG. 7 includes photomicrographs of the macular region of the retina ofa human volunteer subject. Image a is an image obtained by measuring thereflection of white light (standard fundus image). Images b and c aretypical retinal lipofuscin fluorescence images, or pixel intensity maps,with image b showing a lipofuscin fluorescence digital fundus imageobtained under 488 nm excitation and image c showing a lipofuscinfluorescence digital fundus image obtained under 532 nm excitation, eachobtained at near IR wavelengths (λ>700 nm). The field of view for imagea is larger than for images b and c in order to illustrate the relativelocation of the macular region (gray shaded area on left side of imagea) with respect to the optic nerve disk (bright white spot on right sideof image a). Images b and c are centered on the macular region and arerecorded, respectively, with 488 nm light that is absorbed by bothlipofuscin and macular pigments, and with 532 nm light that fallsoutside the absorption range of macular pigments, and therefore onlyweakly excites the lipofuscin emission.

The two images b and c differ substantially in the macular region,showing pronounced absorption due to MP under 488 nm light, andvirtually no absorption under 532 nm light excitation, as expected fromthe MP absorption behavior (see FIG. 1).

Small blood vessel patterns in these images can be used as landmarks toalign these two distributions for digital image processing. A digitalsubtraction image due only to the MP absorption can be obtained bysubtracting image c from image b. For example, the spatial extent of MPand its topographic concentration distribution can be obtained bydigitally subtracting image c, serving as a reference pixel intensitymap, from image b, which has pixel areas with reduced intensities due toabsorption of the lipofuscin emission by MP (central shaded area).

Particularly, the lipofuscin fluorescence intensity map obtained under532 nm excitation can be used to assess the distribution of thelipofuscin and melanin concentration throughout the retinal area ofinterest, i.e. the area centered on the fovea and subtending to about 7degrees or higher eccentricity. This is the case since the I₅₃₂intensities decrease from the macular image location with I_(max,532) tothe peripheral location with I_(min,532) only by the known correctionfactor, c_(Gauss) (this factor is described above and is due to thecombined Gaussian laser excitation and fluorescence response profile).

Next, in order to derive the MP optical density in the fovea orsurrounding regions, The invention use the measured lipofuscinfluorescence intensity maps obtained under 488 nm and 532 nm excitation,respectively, to determine the MP optical density difference fromequation (9), which becomes $\begin{matrix}{{{O.D.} = {1.2 \times \left\lbrack {{\log\left( \frac{\quad{\overset{\_}{I}}_{\max}}{\quad{\overset{\_}{I}}_{\min}} \right)}_{\lambda = 488} - {\log\left( \frac{\quad{\overset{\_}{I}}_{\max}}{\quad{\overset{\_}{I}}_{\min}} \right)}_{\lambda = 532}} \right\rbrack}},} & \left( {{Eq}.\quad 10} \right)\end{matrix}$where {overscore (I)}_(max) and {overscore (I)}_(min) are intensitiesaveraged over certain pixel areas (see below). The factor 1.2 takes intoaccount the ratio between the absorption of MP at its maximum (460 nm)and the used excitation wavelengths of 488 nm and 532 nm. The inventiondetermined this factor from the absorption spectrum of MP measured froman excised eye cup.

FIG. 8 illustrates schematics showing processing of specific CCD pixelintensity regions chosen to derive optical density values of MPabsorption at any desired location in the retina. By way of example, forthe calculation of average MP levels in specific locations or alongnasal-temporal and inferior-superior meridians, individual pixels can begrouped into disks with a diameter of 20 pixels, as illustrated in FIG.8. Averaged peripheral pixel intensities are determined from twelveperipheral pixel disks located on a circle with 7 degrees eccentricityto the fovea, and the average pixel intensity in an area of interest isdetermined from an additional, single, disk positioned in that area, forexample in the fovea, as shown in FIG. 8. One central disk is located atthe center of the macula, the foveola, with a resulting intensityI_(min) (ave). Twelve additional discs are chosen on a circle with 7degrees eccentricity to the foveola, with equidistant spacing, tocalculate an average fluorescence intensity I_(max) (ave) in theperiphery. The maximum MP image contrast, derived from these twoaveraged intensities, is proportional to the optical density of MP inthe center of the macula, according to equation (8) or (10). Disks inbetween the center and the peripheral circle (not shown) can be chosento calculate the image contrast and MP at any eccentricity toward theperipheral retina.

In measurements of dozens of healthy subjects it was seen that imagesrecorded with 532 mn excitation contributed only 5% change to the MPlevels obtained from imaged recorded with 488 nm excitation alone. In apreferred embodiment a single wavelength, 488 nm laser exposure istherefore adequate to determine the MP levels with high (about 95%)accuracy, and for most practical purposes it is therefore not necessaryto use an additional second excitation wavelength outside the MPabsorption range for MP level determinations.

However, in the presence of macular pathology, it is very likely thatdual wavelength measurements will be necessary to account for spatiallynon-uniform lipofuscin and melanin distributions. The fluorescence basedMP detection method, as carried out in this invention, shows astatistically significant correlation with MP measurements using highlyMP specific Resonance Raman spectroscopy in healthy subjects, as seenfrom a direct comparison of both techniques in a subgroup of 48subjects. MP levels showed a tight correlation up to integrated MPoptical density levels of about 0.35. At higher MP levels, deviationsoccur that are likely correlated to nonlinear effects of both opticalmethods at higher MP levels. Possible factors include (a) screeningeffects in the MP Raman and lipofuscin fluorescence responses at highmolecule concentrations, (b) an under-sampling of the MP distributionscaused by lens opacity effects in elderly subjects in the Raman method,(c) non-vanishing MP levels at the peripheral retinal reference point inthe fluorescence based method, and (d) residual absorption ofphotoreceptors in the fluorescence detection. The invention expectsfurther insight into the differences between both methods by a directcomparison of fluorescence based imaging results with Raman images of MPdistributions in the same subjects.

The invention can be used in a variety of contexts to benefit subjects.For example, correlative studies can be performed using spatial imagingof MP concentrations with changes over time or particular shapes (SeeFIGS. 14 and 16) to correlate MP levels with pathologies, and potentialincreases occurring upon dietary interventions and supplementation.Particularly, either 3d patterns or observed changes with aging may beindicative of certain diseases. In a preferred aspect of the invention,the measured MP data can be used to proscribe a treatment, vitamins,dietary supplements, etc. to a subject. Because the test can beperformed in under a second using low light power, even LEDs in someinstances, and in non-mydriatic conditions, the test is ideal for rapidand proscribed or elective testing in a variety of environments, fromdoctors offices and hospitals to less formal setting such as optometryshops and dietary supplement stores. The data obtained from a test canbe used to customize treatment plans and counteract health problems.

The following examples are given to illustrate the present invention,and are not intended to limit the scope of the invention.

EXAMPLES Example 1

For the determination of the MP optical density, the obtained imagecontrast is important, being directly proportional to the MP opticaldensity. In living eyes, care must be taken that the lipofuscinfluorescence image contrast is not artifactually reduced or enhanced byother absorbing or fluorescing compounds besides MP. For example, if anartifactual fluorescence signal existed in the macula, it would add tothe fluorescence level of lipofuscin, and thus reduce the imagecontrast. Likewise, an extra fluorescence signal in the periphery, or anabsorption in the center from unbleached photoreceptors, would enhancethe image contrast. A presence of artifacts would be obvious if therewere to be a wavelength dependence of the obtained image contrast, i.e.if the image contrast were to change depending on which spectralportions of the lipofuscin fluorescence were included in the measurementand subsequent derivation of the MP levels.

With reference therefore to FIG. 10, in order to check for the existenceof artifacts, a series of measurements were carried out in which thedetected lipofuscin fluorescence signals were limited to progressivelylonger wavelength regions. Using long-wavelength step functiontransmission filters with successively longer cut-on wavelengths withthe two-wavelength measuring scheme (488 and 532 nm), and testing theeffect on three subjects, the MP results shown in FIG. 10 were obtained.The optical density values were computed with equation (10), andincluded correction of the respective digital images for laser intensityvariations within the excitation disk (c_(Gauss)). Enhancement factorsof 1.71, 1.72, and 1.85 were observed, respectively, of the imagecontrast at wavelengths limited to 700 nm and beyond, as compared to MPlevels obtained when including fluorescence components in the blue-greenspectral region. The variability of the results in FIG. 10 did notcorrelate with the age of the subjects.

Example 2

To test whether the interfering green fluorescence component originatesfrom intrinsic MP fluorescence (see FIG. 1 for the emission spectrum),an excised eye cup was imaged from a donor eye, using essentially thesame imaging setup as for living eyes an MP O.D. value of about 0.4±10%was obtained for this sample throughout the wavelength range 530-700 nm,thus revealing an absence of a wavelength effect on the optical density.This proves that intrinsic MP fluorescence is too weak to interfere withthe obtainable image contrast of the lipofuscin fluorescence basedimaging technique.

Example 3

Next, the wavelength dependence for the foveal MP levels of a subjecthaving an implanted, non-fluorescing prosthetic eye lens was measured.Again, an absence of a wavelength effect over the range 530-700 nm wasobserved. From these results it is seen that the artifactual greenfluorescence present in living eyes originates from fluorophores presentin the living eye lens. For the purpose of obtaining optimum imagecontrast and corresponding MP level determination, according to theinvention it is important to detect fluorescence levels only on the longwavelength shoulder of the lipofuscin fluorescence, i.e. above about 700nm.

Example 4

In order to test the setup in FIG. 4 and the image processing routines,a tissue phantom was imaged in exactly the same geometry and under thesame conditions as used for living eye measurements (180 degreedetection geometry with CCD camera detection), including identical lightfiltering and pixel processing routines. The tissue phantom consisted ofa dried drop of lutein solution spotted onto one of the side windows ofa thin-walled cuvette filled with a methanolic A2E solution, asillustrated in FIGS. 9 a and 9 b. The optical density of the solutionhad a value of 0.35 at 488 nm. The dried lutein spot was roughlycircular in diameter (about 2.5 mm), and had a non-uniform thickness andconcentration that increased from the center on outwards. Using a smalldiameter (200 μm) 488 nm laser excitation spot, the lipofuscinfluorescence intensity of the lutein spot/A2E solution combination, orthe A2E solution alone, was imaged progressing from the center of thelutein spot toward the periphery and beyond. Simultaneously, the in-situsample transmissions at the laser wavelength was also measured, using alight chopper in the excitation laser beam, and phase-sensitivedetection of the transmitted laser beam with a silicon photo-detectorplaced behind the tissue phantom. From these measurementslipofuscin-emission based optical densities of local luteinconcentrations inside the phantom spot was correlated with opticaldensity values obtained from transmission data. The results aredisplayed in FIG. 9 c for a few dozen locations within the lutein spot.They demonstrate excellent agreement between the two methods(correlation coefficient R=0.96), proving that the imaging proceduresand data processing routines lead indeed to the desired lutein opticaldensity determination for this tissue phantom. Transmission basedabsorption levels had an accuracy of 2%; indirect, lipofuscin basedabsorption levels an accuracy of 4%.

Examples 5-8

FIG. 11 shows representative MP imaging results for four healthyvolunteer subjects, labeled (a)-(d), with the obtained spatial MPdistributions displayed as gray-scaled intensity levels with 16-bitaccuracy. FIG. 11 is lipofuscin fluorescence images of four volunteersubjects (a-d), obtained under 488 nm excitation, shown in gray scale.Fluorescence intensities are lowest in central dark image regions due toabsorption of excitation light by MP. Note pronounced variation of MPdistributions regarding strength, symmetry, and spatial extent amongindividuals. These tests were carried out with 488 and 532 nm imagingand corrected for spatial laser intensity variation over the excitationdisk (c_(Gauss)). In FIG. 12 intensity line plots are displayed that arederived from the gray-scale images for each subject along nasal-temporaland inferior-superior meridians, both running through the center of themacula, respectively. Plots are derived from pixel intensity maps ofFIG. 10 for each of four subjects (a)-(d). A transmission value in thefoveola can be calculated from the averaged pixel intensities in theperipheral macula and the foveola. In FIG. 13, the MP optical densitydata are plotted for each subject along the nasal-temporal meridian. Inthe line plots of FIGS. 12 and 13, pixel intensities were averaged over14 pixels (280 μm) oriented perpendicularly to the respective meridians,and transmission values were calculated versus distance from the fovea.For one of the subjects, the MP distribution was imaged eight times overa period of four weeks, showing that the MP optical densities could bedetermined with a test-retest accuracy (standard deviation) of 2.4%.

FIG. 14 displays pseudo-color scaled, 3-d MP distributions derived fromthe 2-d gray scaled lipofuscin fluorescence pixel intensity maps of FIG.8. Note significant inter-subject variations in MP levels, symmetries,and spatial extent.

As can be clearly seen from FIGS. 11-14, the spatial widths, symmetries,and concentrations of MP vary significantly between the four subjects.In subject (a), the MP distribution shows only a weakly enhanced centrallevel compared to the parafovea. In subject (b) the MP distribution hasa very low central level and is surrounded by a ring of MP. Subject (c)has a strongly peaked MP distribution in the center, and almost noparafoveal levels and finally, subject (d) has both high central andparafoveal MP levels.

Example 9

To further evaluate the lipofuscin fluorescence-based MP imagingtechnique, a clinical population study was performed involving 70healthy volunteer subjects The demographic characteristics of thepatient population are shown in Table 1 below. TABLE 1 Demographics ofPopulation Number of Normal Subjects 70 Age (yrs: mean +/− SD 53 +/− 16Age Range (yrs) 23-89 Female (%) 50 Male (%) 50 White Subjects (%) 90Nonwhite subjects (%) 10 Nonsmokers (%) 86 Active Smokers (%) 14

The MP measurements of the population sample involving 70 healthysubjects reveal distinct patterns of MP distributions, such as a patternwith high central MP levels surrounded by eccentric shoulders or ringsof lower MP levels. Also, it is seen that MP levels can differ verystrongly among individuals, i.e. by more than an order of magnitude, andthat the average MP levels decline slowly with age in the sampledpopulation (on average by a factor of three between age 25 and age 80).

FIG. 16 shows distinctive MP distribution patterns observed in thesesubjects. Five categories, A-E, based on striking spatial features inthe MP distributions are depicted at the top of FIG. 16. Two examplesare shown for, each category. The bottom of FIG. 16 b illustrates thedistribution of 122 measured eyes among categories A to E. A largefraction of subjects, 28%, has a sharp, central MP distribution. Incategory A, MP optical densities were very low (smaller than about0.05). This is observed for 11% of the population sample. In category B,the MP distribution is laterally extended and has enhanced central MPlevels; it is seen in 22% of the subjects. Category C features only asharp central MP distribution, and is seen in 28% of subjects. CategoryD has a sharp central MP distribution and an additional MP ring locatedin the parafovea; this pattern is seen in 17% of the subjects, and hasbeen previously noted by other investigators using autofluorescence andreflectometry. Finally, category E has a relatively uniform, laterallyextended, MP distribution with no elevated central MP levels, and isseen in 12% of the subjects.

For twenty of the subjects, the invention recorded images for both 488nm and 532 nm excitation, and evaluated the MP levels according toequation (10). However, in all subjects an image contrast due to macularMP concentrations was practically absent under 532 nm excitation. Theimages all looked very similar to that shown in FIG. 7, and contributedat most 5% of the contrast relative to that observed with 488 nmexcitation alone. From this, it was concluded that 532 nm images are notessential for MP determinations, at least not in healthy subjects. Tofacilitate rapid scanning of a clinical population sample, only singlewavelength, 488 nm, measurements were used for the remaining subjects.It was also determined, however, that two-wavelength imaging is requiredto achieve highest accuracies, and it is very likely to be required alsofor subjects with pathologies.

In FIG. 17 illustrates the macular pigment levels measured in 90 eyes ofa clinical population of 70 volunteer subjects, displayed as a functionof age. Each data point corresponds to the maximum MP level determinedfrom lipofuscin fluorescence images over its foveolar region (150 μmdiameter). Filled circles represent subjects measured after cataractsurgery (lens implant). Note the significant variation of MP levelsbetween individuals at any age and the average decline of levels withage. In several subjects with age near 40 years, for example, the MPlevels are seen to range from an O.D. near zero to O.D. of about 0.6.Also, there is a statistically significant decline of average MP levelswith increasing age; with a correlation coefficient r=−0.47. Calculatingthe correlation coefficient with 1 data point per subject (one eye persubject), r=−0.54) was obtained.

Example 10

The correlation of the indirect, lipofuscin fluorescence-based MPdetection technique, was compared with the direct technique of integralResonance Raman detection, the latter measuring MP responses over anapproximately 1.2 mm diameter excitation disk in an integral fashion.For this purpose a clinical population subgroup of 48 subjects wasrecruited, and 72 eyes were measured. The results are shown in FIG. 18,revealing a statistically significant and strong correlation betweenboth methods (correlation coefficient r=0.73, p<0.0001 including alleyes measured, and r=0.75, p<0.0001, including only one eye persubject). The plotted fluorescence based MP optical density levels aremaximum levels at the foveola. They are calculated from lipofuscinfluorescence pixel intensity maps integrated and averaged for a circulararea, centered at the fovea, having 20 pixel diameter (150 μm). Filledcircles in FIG. 18 correspond to data points for subjects with lensimplants. Limiting the correlation to MP optical densities below about0.35, the correlation is even stronger. At optical densities above about0.35, the variance of the levels increases, likely caused by breakdownof the assumptions underlying the lipofuscin based MP detection methodand the resonance Raman methods.

Raman based MP levels are derived from Raman scattered light intensitiesat the carbon double stretch frequency of 1525 cm⁻¹. Since the latterare obtained with a 1 mm diameter, 488 nm laser, excitation spotcentered on the macula, Raman based MP levels are averaged over anapproximately 1 mm diameter area. Raman based levels are not correctedfor media opacities, while fluorescence based levels are not influencedby media opacities. The high correlation therefore demonstratesindirectly that media opacities are not very significant in Ramanmeasurements.

Example 11

To investigate the influence of blood vessels on the spatial MPasymmetries, the measured pixel intensity maps were processed withreduced vertical pixel averaging, and with software filter masks toenhance finer spatial details. Two gray scale images and correspondingnasal-temporal meridional line plots illustrate the results. They areshown in FIG. 15. In the image shown at the top of FIG. 15, the pixelintensity map is unfiltered. In the image shown at the bottom, the mapis filtered and weighted with a Gaussian mask. For both images,resulting transmission line plots are shown resulting from averagingover 15 pixels (solid curves) and 5 pixels (dashed curves),respectively. Averaging of 15 vertical pixels produces smooth curves butreveals pronounced asymmetries in the shoulders of the distribution atabout 400 μm eccentricity. Averaging of only 5 pixels, results in aclearly visible spatial fine structure (about 50 μm width) in theshoulders of the MP distribution, as evidenced by large amplitudeoscillations in the profile in the about 300-600 μm eccentricity range.The overall spatial asymmetry of the profile, however, is not influencedby the blood vessels. In the higher resolution plots, other spatialdetails not related to blood vessels are discernible, too, such as thesmall but clearly resolved dip of the MP absorption profile in thefoveola of this subject.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

1. A method for measuring macular pigments, comprising: illuminating themacular tissue of an eye with light at a wavelength that overlaps theabsorption band of macular carotenoids and the absorption band oflipofuscin, quantifying the light emission from each of the macula andperipheral retina of the eye at an excitation wavelength that liesoutside the macular pigment absorption range and outside the excitationrange of interfering fluorophores, and determining the spatial extentand topographic concentration distribution of macular pigments in themacular tissue from the differing light intensities in the macula andperipheral retina.
 2. A method as defined in claim 1, wherein the act ofilluminating the macula and peripheral retina of an eye with light isperformed with light at a wavelength of from about 450 nm to about 550nm at an exposure on the eye below about 10 mJ/cm² and for less thanabout 1 second.
 3. A method as defined in claim 1, wherein the act ofilluminating the macula and peripheral retina of an eye with lightcomprises illuminating the macula and peripheral retina of an undilatedeye.
 4. A method for diagnosing a subject, comprising: performing amethod as defined in claim 1 upon the eye of a subject, comparing themacular pigment levels to correlative data indicative of one or morepathologies or symptoms, and based upon the comparison, determining thepresence, absence, or degree of one or more pathologies or symptoms. 5.A method for measuring macular pigments, comprising: providing a lightsource that emits light at at least one wavelength that overlaps theabsorption band of macular carotenoids and the absorption band oflipofuscin, directing light from the light source onto macular tissue ofan eye for which macular pigment levels are to be measured, collectinglight emitted from the macular tissue, the collected light comprisinglipofuscin emission from the macular tissue at an excitation wavelengththat lies outside the macular pigment absorption range and outside theexcitation range of interfering fluorophores, quantifying the collectedlight from each of a plurality of locations in the macular tissue andthereby quantifying the lipofuscin emission intensities from each of aplurality of locations, and determining the macular pigment levels inthe macular tissue from the differing lipofuscin emission intensities inthe macula and peripheral retina.
 6. A method as defined in claim 5,wherein the collected light is filtered such that only light above about700 nm is quantified.
 7. A method as defined in claim 5, wherein thelight from the light source has an intensity that does not substantiallyalter macular pigment levels in the macular tissue;
 8. A method asdefined in claim 5, wherein the light source generates coherent light ata wavelength of from about 450 nm to about 550 nm.
 9. A method asdefined in claim 5, wherein the light source generates laser light at awavelength of about 488 nm.
 10. A method as defined in claim 5, whereinthe light source is a light emitting diode.
 11. A method as defined inclaim 5, wherein the light exposure from the light source on the eye isbelow about 10 mJ/cm².
 12. A method as defined in claim 5, wherein thelight exposure from the light source on the eye is for less than about 1second.
 13. A method as defined in claim 5, wherein the collected lightis used to produce digital macular pigment images of the macular tissue.14. A method as defined in claim 5, wherein the lipofuscin emission isfrom fluorescence of the retinal pigment epithelium of the eye uponexcitation with the light from the light source.
 15. A method as definedin claim 14, wherein the fluorescence of the retinal pigment epitheliumis used to produce digital macular pigment images of the macular tissue.16. A method as defined in claim 15, further comprising determiningspatial extent and topographic concentration distribution of the macularpigments by digital image subtraction.
 17. A method for diagnosing asubject, comprising: performing a method as defined in claim 5 upon theeye of the subject; comparing the macular pigment levels to correlativedata indicative of one or more pathologies or symptoms; and based uponthe comparison, determining the presence, absence, or degree of one ormore pathologies or symptoms.
 18. An apparatus for measuring macularpigments, comprising: a light source that generates light at awavelength that is absorbed by macular pigment and lipofuscin and thatproduces an autofluorescence lipofuscin emission; at least one opticalfilter configured for receiving, directly or indirectly, light that isemitted from macular tissue of an eye that has been illuminated withlight from the light source, the optical filter being selective forpassing light at a selected wavelength range such that fluorescencecontributions from ocular media besides the macula are substantiallyblocked; and an optical detector configured for receiving the passedlight from the optical filter and generating a signal indicative of thefluorescence intensities of the lipofuscin emission at the macula andperipheral retina of a subject's eye; and a computing device fordetermining macular pigment concentrations from the fluorescenceintensities of the lipofuscin emission at the macula and peripheralretina.
 19. An apparatus as defined in claim 18, further comprising: oneor more light delivery optical components for directing light from thelight source to a subject's eye; and one or more light collectionoptical components for receiving an autofluorescence lipofuscin emissionfrom the subject's eye and routing the autofluorescence lipofuscinemission from the eye.
 20. An apparatus as defined in claim 18, whereinthe light source comprises a light emitting diode.
 21. An apparatus asdefined in claim 18, wherein the light source generates coherent lightat a wavelength of about 488 nm.
 22. An apparatus as defined in claim18, wherein the optical filter blocks light at wavelengths of less thanabout 700 nm.
 23. An apparatus as defined in claim 18, wherein theoptical detector is selected from the group consisting of a CCD camera,a CCD detector array, an intensified CCD detector array, aphotomultiplier apparatus, and photodiodes.
 24. An apparatus as definedin claim 18, wherein the computing device uses the fluorescenceintensities of the lipofuscin emission to produce digital macularpigment images of the macular tissue.
 25. An apparatus as defined inclaim 18, wherein the computing device uses the fluorescence intensitiesof the lipofuscin emission to obtain spatial extent and topographicconcentration distribution of the macular pigments by digital imagesubtraction.